This chapter is initiated with a general introduction and further surveys reported

literature on marine algae, lectins, and finally focusing more on lectins from

marine algae as indicated below:

1.1 General introduction

1.2 Marine macro algae

1.2a. General characteristics of algae

1.2b Location of algae in India

1.2c. Importance of seaweeds: uses and utilization

1.3 Lectins

Definition of Lectins

1.3a. History of lectins

1.3b. Sources of lectins

1.3c. Characteristics of lectin

1.3d. Biological properties of lectins

1.4 Lectins from marine algae

1.5 Applications of lectins

1

1.1 General introduction

Our earth has enormous resources of natural products which man has been

harnessing since its evolution. Natural products, the secondary or non primary

metabolites produced by terrestrial living organisms have been exploited by

human beings for varied purpose including food, fragrances, pigments,

insecticides and medicines.

Oceans constitute approximately 71 % of earth’s surface. Yet as of today only a

very small fraction of world’s bioactive compound’s supply, comes from the

sea. The ocean offers abundant resources for research and development, yet the

potential of this domain, as a field or new area of marine biotechnology, remains

largely unexplored. However, with the increase in imbalance in human

population the available proportion of land resources have started declining.

Under these conditions, marine environment has evolved as a promising avenue.

Study of marine organisms for their bioactive potential, being an important part

of marine ecosystem, has picked up rhythm in recent years with the growing

recognition of their importance in human life. New trends in drug discovery

from natural sources emphasize on investigation of the marine ecosystem to

explore numerous, complex and novel chemical entities. The selection of

samples for assays of biological activities useable in drug development is often

based on ecological observations and includes specimens with unique (usually

chemical) mechanisms for coping with environmental pressures (Haefner,

2003).

2

The importance of marine organisms as a source of novel bioactive substances is

growing rapidly. Marine organisms represent a valuable source of new

compounds. The biodiversity of the marine environment and the associated

chemical diversity constitutes a practically unlimited resource of new active

substances for the development of bioactive products. Marine organisms are rich

sources of structurally diverse bioactive compounds with various biological

activities. In their review, Aneiros and Garateix, (2004) postulated that with

marine species comprising approximately one half of the total global

biodiversity, the sea offers an enormous resource for novel compounds and can

be classified as the largest remaining reservoir of natural molecules to be

evaluated for drug activity. Moreover, since marine organisms live in very

extreme, competitive and aggressive surroundings, very different in many

aspects from the terrestrial environment, very different kinds of substances can

be identified because they are in a situation that demands the production of quite

specific and potent active molecules. Marine environment serves thus as a

source of functional materials, including polyunsaturated fatty acids (PUFA),

polysaccharides, minerals and vitamins, antioxidants, enzymes and bioactive

peptides (Kim and Wijesekara, 2010).

Seaweeds are macroscopic algae found attached to the bottom in relatively

shallow coastal waters. They grow in the intertidal, shallow and deep-sea areas

up to 180 meter depth and also in estuaries and back-waters on the solid

substratum such as rocks, dead corals, pebbles, shells and other plant material

(Smith, 1944).

3

Global utilization of macroalgae is a multi-billion dollar industry. Much of this

is based on farming of edible species or in the production of agar, carrageen and

alginate. Of all seaweed products, hydrocolloids have had the biggest influence

on modern Western societies (Smit, 2004). They have attained commercial

significance through their use in various industries which exploit their physical

properties such as gelling, water-retention and their ability to emulsify (Renn,

1997). Little commercial exploitation of products extracted from seaweeds

occurs outside the hydrocolloid industry. However, in recent years

pharmaceutical firms have started looking towards marine organisms, including

seaweeds, in their search for new drugs from natural products.

The discovery of the bio-regulatory role of different lectins in the marine algae,

the understanding of the molecular mechanisms of action of some new lectins

obtained from natural sources on specific cellular targets, contributes to consider

algal lectins also as promising lead drug candidates.

Lectins are carbohydrate-binding proteins of non-immune origin which

agglutinate cells or precipitate glycoconjugates (Goldstein et al., 1980). They are

a very heterogeneous group of proteins, artificially classified together, solely on

the basis of their capability to agglutinate cells. The first definition of lectins

was based primarily on the sugar specificity and inhibition of the agglutination

reaction. However, the definition appeared restrictive as it excluded some poorly

agglutinating toxins such as Ricin, Abrin, Modeccin etc, which were known to

contain lectin subunits. Moreover, some lectins contained a second type of

binding site that interacted with non-carbohydrate ligands and lectins were

4

further re-defined as carbohydrate-binding proteins other than antibodies or

enzymes. As a consequence, the presence of at least one non-catalytic domain,

which binds reversibly to a specific carbohydrate, is considered to be the only

criterion for a protein to be considered as a lectin (Peumans and Van Damme,

1995)

Lectins are widely distributed throughout the plant kingdom where they have

been found in a variety of tissues of a large number of different plants as early

as 19th century. Subsequently, lectins have been reported to be present in plant

seeds particularly in cotyledons where they appear during the later stages of

maturation of the seeds (Van damme et al., 1998c).

Many of the animal lectins, even in diverse sources, exhibit sequence similarity

and common features, which serve as a basis for their classification into a

number of families, the most prominent of which are galectins, C-type lectins

and siglecs. Although a number of lectins of various molecular weights have

been found in marine invertebrates, very limited information concerning their

structures has thus far been obtained (Kilpatrick, 2000).

Boyd et al, (1966), first identified a hemagglutinin from marine algae. However,

the first agglutinin to be characterized from marine algae was in 1977 by Rogers

et al., and later several algal hemagglutinins have been isolated, to date.

Unfortunately, the practical uses of marine algal hemagglutinins have been

limited to research and their routine use for clinical purposes remains limited.

5

1.2 Marine macro algae

1.2 a. General characteristics of algae

Seaweeds or benthic marine algae are the group of plants that live either in

marine or brackish water environment on solid substratum such as rocks, dead

corals, pebbles, shells and other plant materials. Seaweeds are macroscopic

algae found attached to the bottom in relatively shallow coastal waters. They

grow in the inter-tidal, shallow and deep sea areas up to 180 meter depth where

0.01% photosynthetic light is available and also in estuaries and back waters

(Smith, 1944).

“Seaweeds” refers to any large, marine benthic algae that are multi-cellular,

macrothallic, and thus differentiated from most algae that are of microscopic

size (Smith, 1944). Like land plants, seaweeds contain photosynthetic pigments

and with the help of sunlight and nutrients in the sea water, they photosynthesize

and produce food. Plant pigments, light exposure, depth, temperature, tides and

shore characteristics combine to create different environments that determine the

distribution and variety among seaweeds.

The important criteria used to distinguish the different algal groups based on the

recent biochemical, physiological and electron microscopic studies are:

photosynthetic pigments, storage food products, cell wall components, fine

structure of cell and flagella (Smith, 1944) Accordingly, marine algae are

classified into three main groups i.e. Chlorophyceae (green algae),

Phaeophyceae (brown algae) and Rhodophyceae (red algae). Like land plants,

6

seaweeds contain photosynthetic pigments and with the help of sunlight and

nutrients present in the seawater, they photosynthesize and produce food.

Seaweeds are similar, in form, with the higher vascular plants but the structure

and function in part significantly differs from that of higher plants. Seaweeds do

not have true roots, stem or leaves. Whole body of the plant is called thallus and

consists of the hold fast, stipe and blade. The hold fast resembles the root to the

higher plants but its function is for attachment and not for nutrient absorption.

The hold fast may be discoidal, rhizoidal, bulbous or branched depending on the

substratum it attaches. The most significant differences of seaweeds from the

higher plant is that their sex organs and sporangia are usually one-celled or if

multi-cellular, their gametes and spores are not enclosed (Prescott, 1984).

1.2b Localization of algae in India

Seaweeds grow abundantly along the coast of India. Rich growth of seaweeds is

found around Tamil Nadu, Gujarat, Lakshwadweep, Andaman and Nicobar

Islands, Mumbai, Ratnagiri, Goa, Karwar and Orissa. Survey carried out by the

Central Salt and Marine and Chemical Research Institute (CSMCRI), Central

Marine Fishery Resource Institute (CMFRI) and other research organizations

have revealed abundant seaweed resources along the coastal belts of South

India. On the west coast, especially in the state of Gujarat, huge seaweed

resource is present on the inter-tidal and sub-tidal regions. These resources

translate into great potential for the development of seaweed-based industries in

India. (Dhargalkar and Pereira, 2005)

7

Although, India (08.04–37.06 N and 68.07–97.25 E), a tropical South Asian

country has a stretch of about 8000 km coastline, excluding its island territories

with 2 million km2 Exclusive Economic Zone (EEZ) and nine maritime states

((Rao and Mantri, 2006), Indian seaweed industry suffers from absence of

commercial cultivation practices, lack of infrastructure for commercial

cultivation and absence of policy support. The seaweed flora of India is highly

diversified and comprises mostly of tropical species, but boreal, temperate and

subtropical elements have also been reported (Rao and Mantri 2006). In all, 271

genera and 1153 species of marine algae, including different forms and varieties

have been enumerated till date from the Indian waters (Anon, 2005).

Distribution of species of seaweed in India have been reported to be (Seaweed

cultivation and utilization, Policy paper 22, NAAS) 202 in Gujarat;

Maharashtra, 152 ; Goa, 75; Karnataka, 39; Kerala, 20; Lakshwadeep, 89; Tamil

Nadu ,302; Andhra Pradesh 78; Orissa 1; West Bengal 6 and Andaman &

Nicobar Islands 34. India presently harvests only about 22,000 tonnes of macro-

algae annually, a mere 2.5 per cent, compared to a potential harvest of 870,000

tonnes.

1.2.c. Importance of seaweeds: uses and utilization

1.2.c1. Food:

People do not have very good impression of seaweeds. They think they are just

some stinking, slimy nuisance that washes up on clean sandy beaches. Most

people do not realize how important seaweeds are, both ecologically and

commercially. In reality, seaweeds are crucial primary producers in oceanic

food webs. They are also valuable sources of food, micronutrients, and raw

8

materials for the pharmaceutical industry. Seaweeds have plenty of essential

nutrients, especially trace elements and several other bioactive substances and

thus, today, seaweeds are considered as the food supplement for 21st century,

and a rich source of proteins, lipids, polysaccharides, mineral, vitamins, and

enzymes.

Products extracted from seaweeds are increasingly being used in medical and

biochemical research. Prior to the 1950s, the medicinal properties of seaweeds

were restricted to traditional and folk medicines (Lincoln et al., 1991). During

the 1980s and 90s, compounds with biological activities or pharmacological

properties (bioactivities) were discovered in marine algae (Mayer & Lehmann,

2000).

Seaweeds are the only source of phytochemicals namely agar-agar, carrageenan

and algin, which are extensively used in various industries such as food,

confectionary, textiles, pharmaceuticals, dairy and paper industries mostly as

gelling, stabilizing and thickening agents.

Seaweeds are used in many maritime countries as a source of food, for industrial

applications and as a fertilizer. The major utilization of these plants as food is in

Asia, particularly Japan, Korea and China, where seaweed cultivation has

become a major industry. In most Western countries, food and animal

consumption of seaweeds is restricted and there has not been any major pressure

to develop seaweed cultivation techniques, even through there is an increasing

tendency to consume seaweeds as “heath food”.

9

1.2c2. Medicinal use of seaweeds

Algae have been the source of about 35% of the newly discovered chemicals

between 1977-1987 followed by sponges (29%) and cnidarians (22%) with

varied functions and applications in medicine, from therapy to research (Corgiat,

1993) as briefly described below. Several seaweeds from the marine ecosystem

have also been reported to have bioactive compounds. For example, Caulerpa,

Carollina, Hypnea, Padina and Sargassum have shown high level of anti-viral

activity while maintaining low levels of cytotoxicity (Zhu et al., 2003).

Depsipeptides extracted from Bryopsis sp. were active against Mycobacterium

tuberculosis (Sayed et al., 2000). Many toxins with excitatory & cytotoxic

activity have been reported from Digenea sp., Chondria sp., Amansia sp. and

other macroalgae. Poly-halogenated monoterpenes, aplysiaterpenoid & relfairine

isolated from Plocamium showed activity against Anopheles larvae and Culex

larvae (Watanabe et al., 1990). Oxylipins isolated from alga had shown activity

resembling eicosanoid hormones which help in innate immunity (Bouarab et al.,

2004). Algal products such as algin, carrageenan, funoran, fucoidan, laminarin,

porphyran and ulvan have been noted to produce hypocholesterolemic and

hypolipidemic responses due to reduced cholesterol absorption in the gut. An

antiplasmic inhibitor has been isolated from Ecklonia karome (Fakuyama et al.,

1989).

Due to its immense potential, drug discovery has been widely encouraged by

funding authorities and a large number of labs are actively in the process of

screening large numbers of pure organic compounds or crude extracts to provide

new leads. Marine algae have historically been an exceptionally rich source of

10

pharmacologically active metabolites with anti-neoplastic, antimicrobial and

antiviral effects (Faulkner, 2000; Tziveleka et al., 2003). Random screenings

were effective to have found marine algae with various biological activities

(Harada et al., 1997), and many of these reports have been reviewed

(Nekhoroshev, 1996). In addition, some natural products previously ascribed to

marine invertebrate animals were proved to be secondary metabolites from algae

(Scheuer, 1990).

Anti-tumor activity is one of the most important aspect in/of marine drugs, and

there are many reports (Fuller et al., 1994; Harada et al., 2002; Mayer and

Gustafson, 2003; Sheu et al., 1997) demonstrating potent cytotoxic functions of

algae and their metabolites. These metabolites have played an important role in

paving the path to new pharmaceutical compounds for anti-tumor drugs (Yoo et

al., 2002). Several representative anti-tumor compounds from algae, such as

Halomon, had been developed upto the clinical phase (Egorin et al., 1996). The

accumulated metabolites have shown various potential biological activities

including antibacterial, antioxidant and α-glucosidase inhibitor activities (Choi

et al., 2000).

1.3 Lectins

Definition of Lectins

Lectins were first described in 1888 by Stillmark, working with castor bean

extracts. Since lectins were originally isolated only from plant extracts and were

used for agglutination of red blood cells, they were also called as

‘phytohemagglutinins’. Later it was reported that they could also be obtained

11

from animal organs, especially those of invertebrates and did not all bind to

erythrocytes. So, in 1954, the term ‘lectin’ was coined (Boyd & Shapleigh,

1959).

International Union of Biochemistry Nomenclature (Dixon, 1981) defined

“lectin” as carbohydrate-binding protein of non-immune origin that agglutinates

cells and precipitates polysaccharides. The emphasis on non-immune origin of

lectins is to distinguish them from carbohydrate-specific antibodies. To stress

that lectins are different from carbohydrate-specific enzymes, such as kinases,

glycosidases, transferases and transporters, which in rare cases agglutinate cells,

another definition has been proposed. By this definition lectins are

carbohydrate-binding proteins that do not modify the carbohydrates to which

they bind (Kocoureck & Horejsi, 1981). It should be noted that in addition to

carbohydrate-binding  sites, lectins may contain one or more sites that interact

with non-carbohydrate ligands (Barondes; 1988).

Carbohydrate-binding proteins include enzymes that act on sugars as substrates,

carbohydrate-specific antibodies, membrane transport proteins involved in sugar

transport and chemotactic detection. The distinct function served by these

proteins is reflected in differences in their intrinsic affinities for

monosacharides, which are often much higher than that of lectins and in their

relative importance of oligosaccharides as ligands, which form the primary high

affinity ligands for lectins.

Of the hundreds of monosaccharides found in nature, large majority of lectins

recognize just a few, primarily mannose, glucose, galactose, N-

12

acetylglucosamine, N-acetylgalactoseamine, fucose and N-acetylneuraminic

acid; in addition, they combine specifically with large numbers of

oligosaccharides composed of these monosaccharides. A striking feature of the

above monosaccharides is that they are typical constituents of animal

glycoconjugates and are present on the surface of cells, including erythrocytes.

Lectins specific for other sugars have very rarely been encountered. This may be

a reflection of the method routinely employed for the detection of lectins,

namely agglutination of erythrocytes, or hemagglutination in brief (Ahmed,

2005).

Lectins differ from antibodies in several different aspects. Many lectins are

found in plants, microorganisms and viruses that are not capable of an immune

response. Another marked difference between the two classes of protein is that

antibodies are structurally similar whereas lectins are structurally diverse. In

general, lectins are oligomeric proteins composed of subunits, one or more of

which carries a sugar-binding site. They vary, however, in size, amino acid

composition, metal requirement, domain organization, subunit number and

assembly, as well as their three dimensional structure and in the constitution of

their binding sites. In their structural diversity, lectins are akin to enzymes,

although they are devoid of catalytic activity. In spite of this variation, they can

be grouped in families of homologous proteins, the largest and best

characterized of which is that of legume lectins (Ahmed, H.; 2005).  

1.3a. History of lectins

Lectins were first designated as “hemagglutinins”, or more commonly as

“phytohemagglutinins”, because they were detected by agglutination of

13

erythrocytes and were found almost exclusively in plants. The first report on the

occurrence in plants of such proteins appeared in 1888 in the doctoral thesis of

Hermann Stillmark, a student of Robert Kobert at the University of Dorpat (now

Tartu) in Estonia. Sillmark was studying the toxicity of the beans of the castor

tree (Ricinus communis). Mixing an extract of beans with blood, he made the

starting observation that erythrocytes were agglutinated. Using the very

primitive methods available at the time, designated by him as “the way of

pharamacological isolation”, namely salt extraction of the beans, precipitation

with magnesium sulphate and ammonium sulphate and dialysis, he obtained “an

odorless, snow-white powder”, which was hemagglutinating (Franz, 1988). It

took however, more than half a century before it was definitely demonstrated

that Stillmark’s “Ricin” was a mixture of a weakly agglutinating protein toxin

and the nontoxic agglutinin (Ricinus communis agglutinin, or RCA). Ricin came

to the attention of the general public in 1978, following its use as a weapon in

the notorious, politically motivated “umbrella murder” (Knight,B. 1979).

Shortly after Stillmark presented his thesis, H. Hellin, another student of Kobert,

discovered that the toxic extract of the Jequirity bean (Abrus precatorious) also

caused the red cells to clump. The new agglutinin was named “abrin”.

Already the early results obtained by Stillmark indicated some selectivity in the

lectin-mediated agglutination of red blood cells from different animals. This

observation was corroborated and further extended by Karl Landsteiner to his

discovery in 1900 of the human A, B and O blood groups. In 1908, Karl

Landsteiner& H. Raubitschek demonstrated that the relative hemagglutinating

14

activities of various seed extracts were quite different when tested with red

blood cells from different animals.

From the commencement, research of lectins has covered/crossed many

milestones as condensed by Sharon and Lis, (2003) in Table 1.1.

Table 1.1- Milestones of lectin research

Source: - Sharon and Lis, 2003, Lectins Second Eds

15

For the first seven or eight decades from the description at the turn of the 19th

century, lectins were hardly of any interest and just a few, almost all from plant,

were investigated in detail. The importance of this early work has only recently

become appreciated, as is clear that studies of plant lectins have helped to

catapult the field of glycobiology in modern era and have made an enormous

contribution to modern biochemistry (Varki et al 1999).

1.3 b. Sources of lectins

Lectins are ubiquitous in nature, and are found in all classes of organisms and

families of organisms although not necessary in every genus or species (Sharon

and Lis, 2003) .They are easy to detect and often to isolate. In addition, some are

available from commercial suppliers. Their tissue and cellular distribution is

variable, and it may be affected by miscellaneous factors, such as development

stage, age and pathological conditions.

1.3b1. Lectins from microorganisms Almost all microorganisms express surface-exposed carbohydrates. The

carbohydrates may be covalently bound, as in glycosylated teichoic acids to

peptidoglycan, or non-covalently bound, as in capsular polysaccharides. Every

surface-exposed polysaccharide is a potential lectin-reactive site. The ability of

lectins to complex with microbial glyco-conjugates has made it possible to

employ the proteins as probes and sorbents for whole cells, mutants and

numerous cellular constituents and metabolites. Microbial receptors for lectins

consist of several unique chemical structures. For example, secreted and often

cell adherent dextrans produced by members of the genera Leuconostoc and

16

Streptococcus are neutral polysaccharides capable of interacting with

Concanvalin A (Con A). Another Con A receptor found on many Gram-positive

bacterial surfaces is glycosylated teichoic acid, a polyelectrolyte.

Many bacterial species and genera express lectins, frequently of more than one

type and with distinct specificity (Ofek & Doyle, 1994; Sharon & Lis, 1997). It

is not known, however, whether individual cells co-express multiple lectins or if

each lectin is confined to a distinct cell population. In Gram negative bacteria

(such as E.coli, K. pneumoniae and Salmonellae spp.), lectins are often in the

form of sub-microscopic hair-like appendages, known as fimbriae (or pili), that

protrude from the surface of the cells. Fimbrial surface lectins are also produced

by Gram positive bacteria, among them the oral Actinomyces naeslundii and

Actinomyces viscosus. Non-fimbrial lectins associated with the bacterial surface

have been purified from Rhizobium lupinii, and Agrobacterium tumefaciens,

also a member of the Rhizobia family (Sharon and Lis, 2003).

Marine surfaces are colonized by a diversity of microorganisms and sessile

marine organisms are collectively known as biofouling communities. Biofouling

process is initiated by the attachment of bacteria to a surface followed by the

settlement and adherence of diatoms, free-swimming algal spores and

invertebrate larvae (Bryers & Characklis; 1982). Some sessile higher organisms

employ chemical defenses against biofouling through the production of

secondary metabolites that inhibit the development and formation of a

biofouling community (Harrison; 1992). For example, furanones produced by

the red alga Delisea pulchra have been reported to inhibit the settlement of

17

common fouling organisms. For marine organisms without intrinsic defense

mechanisms, it has been proposed that protection against fouling is maintained

by the secondary metabolites produced by surface-associated bacteria (Egan et

al., 2001).

A lectin has also been identified in protozoa, (Ward, 1997) a surface protein, in

the pathogenic ameba, Entamoeba histolytica having specificity for Gal/GalNAc

(Petri & Schnaar, 1995). Two lectins, one specific for N-acetylneuramic acid,

the other for N-acetylglucosamine, were isolated from merozoides of the human

malarial parasite Plasmodium flaciparum (Ward, 1997).

1.3b2. Viral lectins

Viruses contain sugar-specific surface proteins or glycoproteins that act as

hemagglutinants and therefore classified as lectins (Sharon & Lis, 1997). Much

information is available on the influenza and polyoma viruses, belonging to the

orthomyxoviruses and papoviruses, respectively. Similar lectins that are less

well defined are found in myxoviruses, such as those of Newcastle disease,

Sendai and rotaviruses. Other viral lectins include those of HIV (Haidar et al.,

1992) and foot-and-mouth diseases (Fry et al., 1999).

1.3b3. Lectins from Fungi

The first lectin to be purified from these sources was from the fruiting bodies of

the meadow mushroom, Agaricus campestris, and the common (commercial)

mushroom, Agaricus bisporus (Goldstein & Poretz, 1986) and now, many other

fungal lectins are known (Guillot & Konska, 1997). Lectins have also been

18

found in phytopathogenic fungi, such as Botrytis cinerea (Kallens et al.1992),

Pleurotus ostreatus (Chattopadhyay et al. 1999; Wang et al. 2000), Rhizoctonia

solani (Candy et al. 2001) in different members of the Sclerotiniaceae

(Goldstein, 1990; Inbar & Chet, 1994) and in the nematode-trapping fungus

Arthrobotrys oligospora (Rosen et al. 1996). Couple of years ago, a lectin with

unique carbohydrate-binding properties, including blood group B specificity,

and high affinity for Galα3Gal and Galα3Galβ4GlcNAc has been isolated from

the mushroom Marasmius oreades (Winter et al. 2002).

Lectins have been isolated from a few yeast species, namely a galactose-specific

one from a fatty acid auxotroph of Saccharomyces cerevisiae (Kundu et al.,

1987) and two from the culture medium of Kluyveromyces bulgaricus, one

specific for galactose and the other for N-acetylglucosamine (Al-Mahmood et al.

1991).

There are few reviews about fungal and mushroom lectins (Singh et al., 2010;

Khan and Khan, 2011). In the last few years, mushroom and other fungal lectins

have attracted wide attention due to their biomedical applications. Singh et al.,

(2011) have recently purified a mucin-specific lectin from Aspergillus nidulans.

Lectins from fungi have previously been reported by Bhowal et al., (2005),

Thakur et al., (2007) Khan et al (2007) and Singh et al., (2010b).

1.3b4. Lectins in plants

Lectins have been detected in over a thousand species of plants and several

hundreds have been isolated (Van Damme et al. 1998a). Some of the better

19

characterized plant lectins and their specificities are listed by Sharon and Lis,

(2003) in Table 1.2. The majority of plant lectins have been isolated from seeds,

especially those of the dicotyledonous legumes, where they accumulate during

maturation and disappear upon germination (Sharon and Lis, 2003). Their

location within the seeds however differs among various plant families

(Rudigers, 1998).

Table 1.2- Characterized plant lectins and their Specificity

Family and species Name/

abbreviation

Location

in plant

Specificity

Monocotyledons

Amaryllidaceae

Galanthus nivalis (snowdrop) GNA Bulb Man

Narcissus pseudonarcissus (daffodil) NPL Bulb Man

Gramineae

Oryza sativa (rice) Seed GlcNac

Salt-stresses Oryza sativa (rice) Seed Man

Triticum aestivum (bread wheat) WGA Germ GlcNac &

NAc & Man

Iridaceae

Iris hollandica (Dutch iris) Bulb Gal/GalNAc

&Man

Liliaceae

Allium sativum (Garlic) ASA Bulb Man

20

Scilla campanulata SCA Bulb Man

Dicotyledons

Caprifoliaceae

Sambucus nigra (elderberry)

SNA Bark Neu5Ac-OS

Compositae

Helianthus tuberosus (Jerusalem)

HTL Tuber Man

Convolvulaceae

Calystegia sepium (hedge bindweed)

Calsepa Rhizome Man &

maltose

Cucurbitaceae

Momordica charanita (bitter gourd)

Seed Gal/GalNAc

Euphorbiaceae

Ricinus communis (castor bean) RCA Seed Gal/GalNAc

Ricin Seed Gal/GalNAc

Leguminosae

Abrus precatotorius (jequirity bean) Abrin Seed Gal/GalNAc

Arachis hypogaea (peanut) PNA Seed Gal/GalNAc

Canavalia ensiformis (jack bean) Con A Seed Man/Glc

Dolichos lablab (lablab purpureum) FRIL Seed Man

Glycine max (soyabean) SBA Seed Gal/GalNAc

Lens culinaris (lentil) LCL Seed Man/Glc

Phaseolus vulgaris (red kidney bean) PHA Seed Gal/GalNAc

Phaseolus lunatus (lima bean) LBA Seed Gal/GalNAc

Pisum sativum (pea) PSL Seed Man/Glc

Viscum album (mistletoe) Viscumin Green Gal/GalNAc

21

tissue

Phytolacca Americana (pokeweed) PWM Root (GlcNAc)

Lycopersicon esculentum (tomato) Fruit (GlcNAc)

Solanum tuberosum (potato) STL Tuber (GlcNAc)

(Source:- Sharon and Lis, 2003,Lectins Second edition)

Besides seeds, lectins have been found in all kinds of vegetative tissue. The

level of lectins in these tissues is variable, and exhibits seasonal changes. It is

usually lower than in seeds, but can be as high as 30% of the total tissue

proteins, e.g., the bulb lectins of garlic and ransom, or as low as 0.01% in leaves

of the leek (Peumans et al.2000). Most plant tissues contain a single lectin,

although occasionally two (or more) lectins differing in their sugar specificities

and other properties are found in the same tissue (Peumans et al.2000). The most

extensively studied plant with respect to the distribution of lectins in various

tissues is Dolichos biflorus, wherein the leaves contain a lectin (DB58)

homologous to the seed lectin (DBL), but with some differences in the fine

specificity (Etzler, 1997). In addition, a root lectin (LNP) has been found in the

same plant that is distinct from the lectin in its seed both in amino acid

composition, molecular weight, isoelectric point and specificity (Etlzer et al.

1999).

Lectins isolated in India from plants

There are several reports of lectins isolated from plants in India, some of which

have been purified, characterized and their properties have been assessed. (Islam

et al,2009). D-galactose-binding lectin has been identified from the seeds of the

22

Indian coral tree Erythrina variegata having a leucoagglutinating property

(Datta and Bashu 1981). Soon after in 1985, Khan et al purified a basic lectin

from the seeds of winged bean Psophocarpus tetragonolobus and termed it

WBA l. Using affinity chromatography followed by gel filteration, Dam et al

(1997) purified two mannose-binding lectins. Since these were extracted from

garlic bulbs, they were termed Allium sativum agglutinin, ASAI (25kDa) and

ASAIII (48kDa). Kaur et al (2005) purified an N-acetyl-D-lactosamine-specific

(LacNAc) lectin that was extracted from tubers of Alocasia cucullata and further

purified by affinity chromatography. This lectin was reported to be a potent

mitogen for human mononuclear cells at low concentrations but had growth-

inhibition potential towards cancer cell lines at higher doses. A glucose-specific

lectin was isolated from the roots of Sesbania aculeate (Biswa et al., 2009).

This lectin bound with lipopolysaccharides isolated from different rhizobial

strains indicating the plant’s interaction with multiple rhizobial species.

1.3b5. Lectins identified from Animals

Practically all classes and subclasses of invertebrates examined have lectins.

These includes crabs, snails, worms (helminths) (Greenhalgh et al., 1999;

Hirabayashi et al., 1998), insects (Ingram & Molyneux, 1991; Kubo et al.,

2001), mollusks and sponges (Muller et al. 1997). Lectins in invertebrates are

present mainly in the hemolymph and sexual organs (Vasta, 1992). The best

known invertebrate lectins are from the garden snail, Helix pomatia, from the

body wall of the slug, Limax flavus, and from the serum of the horseshoe crab,

Limulus polyphemus (Kilpatrick, 2000).

23

Many invertebrates contain multiple lectins, several of which have been purified

and characterized. Examples are the four and three lectins, respectively, from

cockroaches, Periplaneta Americana and Blaberus discoidalis, two from sea

urchin, and three of sea cucumber (Cucumaria echinata) (Kilpatrick, 2000)

Although lectins from various plant and invertebrate sources have been known

for many years, their presence in vertebrate tissues has been investigated first by

Ashwell and Morell, (1977). They have described a hepatic-binding protein

which has been implicated in the clearance of glycoproteins from plasma; in

mammals, this binding protein is a β-galactoside-specific, integral membrane

protein of large molecular weight, which can be solubilized by detergents but

not by hapten saccharides and which requires divalent cations for binding

activity. Subsequently, hepatic and reticuloendothelial cell-binding proteins

which recognize mannose, N-acetylglucosamine, and fucose have been detected

(Briles et al, 1979). A "lectin" from platelet plasma membranes has been

described by Gartner et al. (1986) which is inhibited by free amino sugars and

amino acids; the large external transformation-sensitive (LETS) protein or

fibronectin also agglutinates erythrocytes and is inhibited by amines (Yamada,

1975). In addition to these membrane-bound lectins, several soluble lectins have

been identified in vertebrate tissues. Mir-Lechaire and Barondes (1978) have

reported a lectin from chick embryo muscle which is specific for N-

acetylgalactosamine. The most widely occurring family of vertebrate lectins is

that of the galectins, so called because they are glactose specific lectins. Many

mammalian galectins have been described, as well as many additional ones from

24

other species, including birds, lower vertebrates, worms and sponges (Leffler,

2001; Rabinovich et al., 2002).

Lectins isolated from marine organisms in India

Dam et al., (1994) isolated a heparin-binding lectin, Anadarin MS, from the

plasma of the marine clam Anadara granosa. This lectin agglutinated infective

promastigotes of Leishmania donovani suggesting its role as a novel

biochemical surface marker for the parasite. Devaraj et al (1995), isolated and

characterized a high molecular weight glycoprotein from embryonated eggs of

the mole crab, Emerita asiatica. A natural agglutinin was further purified and

characterised from the serum of the hermit crab Diogenes affinis (Murali et al.,

1999). The HA activity of D. affinis agglutinin was susceptible to inhibition by

lipopolysaccharides from diverse Gram-negative bacteria, indicating a possible

In-vivo role of this humoral agglutinin in the host immune response against

bacterial infections. In addition to tissues, a novel lectin was purified from the

coelomic fluid of the sea cucumber Holothuria scabra (HSL) and from the foot

muscle of bivalve Macoma birmanica.

1.3c.Characteristics of lectins Lectins have accordingly been defined as sugar-binding proteins of non-immune

origin that agglutinate cells and precipitate polysaccharides or glycoproteins

(Goldstein et al., 1980). The carbohydrate specificity of lectins has made them

attractive proteins. This property has enabled them to become useful tools for

various scientific purposes including detection and identification of blood

groups and microorganisms, mitogenic stimulation of immune cells,

25

determination of carbohydrates in solutions, on macromolecules and cells,

purification of glycoproteins and cell fractionation and as a tool for taxonomy.

They have also been used as molecular probes for histochemical studies. Griffin

et al. (1995) first demonstrated that Codium fragile lectin could be conjugated to

gold particles and could be used as a histochemical reagent.

Lectins are multivalent i.e. they posses at least two sugar recognition sites which

enable them to agglutinate animal and plant cells and/or to precipitate

polysaccharides, glycoproteins, teichoic acids, glycolipids etc. (Leiner et al.,

1986).

Lectins differ markedly in their sugar-binding specificity. A sequence

participating in carbohydrate binding site of Concanavalin A, for instance, is

poorly conserved in other lectins. However, though termed anti-carbohydrate

antibodies, there are differences such as:

a) Antibody synthesis is inducible whereas lectin synthesis is not.

b) Antibodies can be produced against every determinant, lectins only against a

defined set of sugar molecules.

c) All antibodies are a single class of protein family whereas lectins belong to

different protein families.

d) Some lectins require metal ions as chelating agents while antibodies do not.

e) Antibodies are structurally similar, whereas lectins are subunits, one or more

of which carries a sugar-binding sites.

26

Studies on marine algal lectins reveal a proteinaceous nature similar to those

from land plants, but different in some of their properties. They have generally

lower molecular masses than most land plant lectins and are more specific for

complex oligosaccharides or glycoproteins.

The agglutinating and precipitating activities of lectins are similar to those of

antibodies. They can likewise be specifically inhibited by low molecular weight

compounds (haptens), which in case of lectins are sugars or sugar-containing

ones. Many lectins are found in plants, microorganisms and viruses, which are

not capable of an immune response. Being specific for certain blood groups,

lectins can also prove useful in blood typing, since there is natural non-

availability of anti-O antibodies (Kocourek, 1986). They vary, however, in size,

amino acid composition, metal requirement, domain organization, subunit

number and assembly, as well as in their three dimensional structure and in the

constitution of their combining sites. In their structure diversity, lectins are akin

to enzymes, although they are devoid of catalytic activity (Sharon and Lis,

1990). In spite of this variation, they can be grouped in families of homologous

proteins, the largest and best characterized of which is that of the legume lectins

(Van Damme et al., 1998).

1.3.d. Biological properties of lectins

Some important biological properties of lectins are as follows:-

I. Agglutination of cells is the easiest way to detect lectins. The ability of

lectins ability to agglutinate cells distinguishes them from other sugar

27

binding macromolecules such as glycoside & glycotransferases

(Goldstein et al., 1980).

II. Lectins trigger quiescent, non-dividing cells into a state of proliferation

(Trevin et al., 1986).

III. Mitogenic activity of lectins was first reported from red kidney beans

Phaseolus vulgaris (Nowell, 1960).

IV. Lectins co-stimulate T-cell proliferation along with cytokines (Gollob et

al., 1995).

V. Wheat gram lectin (Kurisu et al., 1980) & few others like Griffonia

simplicifilia (Maddox et al., 1982) posess the ability to mediate

carbohydrate-specific binding of mouse macrophages and tumor cells

and to induce killing of tumor cells by macrophages.

VI. Lectins mediate binding and phagocytosis of target cells (Sharon; 1984).

Thus the binding of Con A to the surface of macrophages mediates the

attachment of bacteria to the macrophages, although no phagocytosis of

bacteria was observed (Allen et al., 1974).

VII. Con A, a wheat gram lectin and some other lectins mimic the effect of

insulin on adipocytes such as stimulation of lipogenesis, transport,

oxidation and inhibition of lypolysis (Shechter et al., 1981).

Insulinomimetic activities were also observed in vitro (Margaret et al.,

2000).

VIII. Cytotoxic lectins have been isolated from an extract of Viscum album.

Tumor cells that was treated with this lectin showed typical apoptotic

cell death, with apparent DNA fragmentation (Ichiro Azuma et al.,

1998).

28

IX. Apoptosis activity was shown to be blocked by the addition of Zn++ or

inhibition by Ca++/Mg++ dependent endonucleases in a dose dependent

manner (Yoon et al., 1998).

X. The lectin designated Hypnin A, from red alga Hypnea japonica

inhibited platelet aggregation in a dose dependent manner (Matsubara et

al., 1996).

Lectins can bind reversibly with free sugars or with sugar residues of

polysaccharides, glycoproteins, or glycolipids (Goldstein and Poretz, 1986).

Both the lectin and its ligand can be free or bound. Lectins can bind to

glycoprotein receptors on cell membranes and this binding is necessary for cell

agglutination.

1.4 Lectins from Marine algae

Marine natural products have attracted the attention of biologists and chemists,

the world over, for the past few decades. As a potential source for new drug

discovery, marine natural products have attracted scientists from different

disciplines such as different branches of chemistry, pharmacology, biology and

ecology. This interest has led to the discovery of almost 8,500 marine natural

products to date and many of the compounds have shown very promising

biological activity (Faulkner et al., 2000). The ocean is also considered to be a

great source of potential drugs (Bhakuni and Ravat, 2005).

The increasing interest in marine natural product’s chemistry has led to the

discovery of new biologically-active compounds and marine algae have been

29

subjected to increasing study for this purpose (Amico, 1995). They have been

reported to contain high amounts of water-soluble macromolecules such as

polysaccharides, proteins, glycoproteins and other less polar compounds of low

molecular weight, some of them exhibiting particular biological properties in

vitro.

Though lectins have been isolated and characterized from various biological

sources, mainly land plants, there is a limited amount of information available

about algal lectins in comparison with those from higher plants and

invertebrates. However, considering their particular characteristics, marine algal

lectins appear to be a potential tool for biochemical and biomedical applications.

Biochemical experiments based on agglutinating tests have revealed the

presence of hemagglutinating activity in many algal extracts against

erythrocytes from several animal species. In most studies this hemagglutinating

activity is referred to the presence of proteins or glycoproteins having

specificities for carbohydrate structures binding selectively to red blood cells

and microorganisms. Compared with lectins from land plants, discovered as way

back as in 1888, the occurrence of lectins from marine algae, however, was first

reported in 1966 by Boyd exhibiting the protein in the sap of some marine algae

(Boyd et al., 1966). Following this pioneering work, other workers have

demonstrated that lectins are present in many algal species (Blunden et al.,

1975; Rogers et al., 1980; Munoz et al., 1987; Hori et al., 1981 & 1988 Dalton

et al., 1995; Freitas et al.,1997; Kakita et al., 1999; Calvete et al., 2000;

Benevides et al., 2001; Sampaio et al., 2002; Wang et al., 2004; Nagano et al.,

2005; Kim et al., 2006; Yoon et al., 2008; Dinh et al., 2009; Han et al., 2010;

30

Molchanova et al., 2010; Jung et al., 2010; Sato et al., 2011). Later workers have

extended this information by studying a wide range of species across the globe.

Marine algae have been screened in surveys at/in Puerto Rico (Boyd et al.,

1966), England (Blunden et al., 1975, 1978; Rogers et al., 1980), Japan (Hori et

al., 1981, 1988), Spain (Fábregas et al., 1985, 1992), United States (Chiles and

Bird, 1989; Bird et al., 1993), Brazil (Ainouz and Sampaio, 1991; Ainouz et al.,

1992; Freitas et al., 1997), Vietnam (Dinh et al., 2009) and Korea (Kim et al.,

2006; Han et al., 2010, Jung et al., 2010). More than 200 algal species have so

far been reported to contain hemagglutinins. The first agglutinin to be isolated

and characterized from marine algae was by Rogers et al., (1977).

Further, most of marine algal lectins do not require divalent cations for their

biological activity (Rogers and Hori, 1993). They occur mainly in monomeric

form and have a high content of acidic amino acids with isoelectric points from

4 to 6. Although several studies on lectins from marine algae have been reported

(Fabregas, 1998; Oliveira et al., 2002), few lectins from algae have been

characterized in detail.

Lectins show differential agglutination property with RBCs isolated from

different sources as well as with differential processing of RBCs. Based on the

hemagglutination properties of lectins, Boyd et al., (1996) and Blunden et al.,

(1975) assayed blood group -specific lectins. In 1994, Dalton et al. investigated

protein extracts from 9 species from green & red marine macro algae for their

ability to agglutinate human blood groups A, B, O, sheep and rabbit

erythrocytes. Chiles and bird (1989) investigated 15 species of algae, of which

31

all showed agglutination against rabbit erythrocytes but only 7 showed

agglutination activity of human erythrocytes. Hori (1990) isolated 12 different

lectins from four species of algae. Of these, four were observed to agglutinate

trypsinized rabbit erythrocytes strongly and their activity was inhibited by

glycoproteins. All specimen studied by Bird et al. (1993) were found to

agglutinate either sheep or rabbit erythrocytes. Trypsinization and Ca++

enhancement of agglutinating lectin activity were shown by Aleli et al. (2000).

The progress in developing applications for algal lectins has been limited mainly

due to three factors. First, very few scientists have been focused on studying

algal lectins. This has led to a paucity of information concerning characteristics

which contribute to their properties In vitro and their possible functions In vivo.

Secondly, marine macro-algae for lectin research are collected from their natural

habitat and thus species which contain interesting lectins may occur in remote

geographical areas or may be relatively rare plants, both of which contribute to

collection difficulties apart from ecological and conservation concerns. Third,

algal extracts have low concentrations of lectins (Rogers et al., 1986), making

detection and production of sufficient purified lectin for detailed biochemical

characterization a difficult endeavor.

The first two of these problems are now being resolved. Many researchers have

made important contributions by their observations on lectins from different

marine algae. The availability of marine algal species known to contain

interesting lectins may be improved by cultivation of relevant species (Rogers et

al., 1982). Also cloning the lectin genes may results in the production of

recombinant lectins.

32

It is in the third area i.e. marine algal lectin detection, purification and

characterization, that most progress has been made (Rogers and Hori, 1993).

Among them, hemagglutinins (lectins) have been isolated from about 50

species. Characterization studies reveal that many algal lectins, especially from

red algae, share the common characteristics of low-molecular size, monomeric

form, having no affinity for monosaccharides and being more specific for

complex oligosaccharides or glycoproteins and thermo stable. Further, most of

marine algal lectins do not require divalent cations for their biological activity

(Hori et al., 1990), indicating that algae are also a good source of new lectins.

They occur mainly in monomeric form and have a high content of acidic amino

acid, with isoelectric points from 4 to 6 (Costa et al., 1999).

The first amino acid sequence of a lectin from marine algae was reported by

Calvete et al., (2000). To date, amino acid sequences of only five marine algae

lectins have been reported. These are from red alga Bryothamnion triquetrum

(Calvete et al., 2000), Hypnea japonica (Hori et al., 2000), Ulva pertusa (Wang

et al., 2004) and Hypnea cervicornis (HCA) and Hypnea musciformis (HML)

(Nagano et al., 2005). Characterization of these lectin genes may help

researchers to further understand the difference between terrestrial plant and

marine algal lectins.

1.5. Applications of lectins

Lectins are widely employed in research for diverse purposes, primarily those in

which detection, identification and functional evaluations of carbohydrates is

33

needed, and are also making a mark on medicine (Gabious et al., 1993;

Goldstein et al., 1997; Rhodes et al., 1998). They offer many advantages,

including ready availability, distinct specificity, and high solubility. Thus, lectin

binding has frequently been used to demonstrate that the membrane receptors

for many hormones, growth factors, neurotransmitters and toxins are

glycoconjugates. Studies with lectins have been largely responsible for the

realization that carbohydrates play a key role in cell-recognition (Sharon and

Lis, 1989; Sharon, 1993), and for expanding the understanding of tissue-bound

carbohydrates in histology and histopathology (Ewen et al., 1998). A new and

promising application is lectin replacement therapy for the treatment of patients

suffering from a lectin deficiency disease (Valdimarsson et al., 1998;

Kilapatrick, 2002b)

In a nut-shell, the varied and major applications of lectins have been

summarized by Sharon and Lis, (2003) as below:

• Cell identification and separation (Yarema and Bertozzi, 2001)

• Detection, isolation and structural studies of glycoproteins

• Investigation of carbohydrates on cells and sub-cellular organelles;

histochemistry and cytochemistry. Selection of lectin-resistant mutants

• Studies of glycoprotein biosynthesis

• Diagnosis and targeting

34

Fig 1.1:- Lectin-based cell-selection strategies ((Yarema and Bertozzi Genome

Biology 2001).

• Mitogenic stimulation of lymphocytes (Kilpartrick, 1998.) Mitogenic

lectins mimic the action of antigens on lymphocytes, except that they

activate a large proportion (as much as 70-80%) of the cells, whereas

antigens stimulate only specific clones. Because of their ability to

stimulate multiple lymphocyte clones, lectins are classified as polyclonal

mitogens.

• Evaluation of immunocompetence

35

Of the many mitogenic lectins, only Concanavalin-A, PHA and PWM are

employed in clinical laboratories as an easy and simple means to assess the

immunocompetence of patients suffering from a diversity of diseases and to

monitor the effects of various immunosuppressive and immunotherapeutic

manipulations (Di Sabato, 1987; Kilpatrick, 1998).

• Karyotyping

• Bone marrow transplantation

• Enzyme replacement therapy

• Construction of Immunotoxins

Attempts are being made to take advantage of the toxicity of ricin for

therapeutic purpose, through the construction of immunotoxins (Ghetie et al.,

2001). These are hybrid molecules, made by covalently linking a toxin (usually

ricin) to monoclonal antibodies against the cells that one wishes to kill. The

antibodies guide the immunotoxin to the target cells, which are then eliminated

by action of the toxin.

Algal lectins also have been studied for their properties..from various biological

functions such as anti-tumor, mitogenic, and anti-virus activities (Hori et al.,

2007). As a practical example, strong anti- HIV lectins have recently been

isolated ((Boyd et al., 1997; Botos et al., 2002; Bewley et al., 2004) and

characterized from blue-green algae (cyanobacteria); Sato et al., 2007) and a red

alga (Mori et al., 2005). Studies performed with extracts of several marine algae

demonstrated that these extracts possess antinociceptive activity (Vieira et al.,

2004; Neves et al., 2007, Figueiredo et al., 2010). Lectins from the Eucheuma serra

36

37

algae are generally obtained in the high yields. ESA-2 shows various biological

activities such as mitogenic activity for mouse and human lymphocytes

(Kawakubo et al., 1997), in vitro growth inhibition of tumor cells (Suzuki et al.,

2000) and antibacterial activity (Liao et al., 2003). Algal lectins play a pivotal

role in cell-cell recognition and thus consequently find application in the fields

of immunology and wound healing process (Kim et al., 2001, 2006, and 2007)

Although considerable progress has been made in understanding the

biochemical character of lectins, little is known about their biological role in

nature. Hori et al. (1988) suggested that lectins may play a common, but as yet

unknown, physiological function in marine algae.

Thus, lectins serve as useful tools and markers in biological research with

unlimited use. Thus, algal lectins are interesting targets for basic research into

lectins and their applications.

Aim

The main objective of the present study was to isolate lectins from untapped marine

algae along the coast of Goa, India. Collection and identification of the marine algae

were the preliminary objective of the present study. Followed by isolation of collected

marine algae and confirmation of hemagglutination activity were the core design of

this study. Selection of species for further purification, biochemical characterizations

of these lectins were studied. This work include the biotechnological potential of the

purified lectins towards cancer cell lines, antimicrobial and antioxidant activity. This

work, therefore, been done with the following objectives in mind.

Objectives of the study

The present study was initiated with the following objectives:

1) To collect and identify marine macro algae commonly found along the shores

of Goa.

2) To screen for prospective hemagglutinins and selection of two species that

show highest activity in terms of agglutination.

3) Extraction of a hemagglutinin from each of the algae selected.

4) Purification of lectin from both the species.

5) Biochemical Characterization of both the lectins.

6) Exploring the potential biotechnological application of the lectin.